1. Field of the invention
This invention is directed to methods for the gel electrophoresis separation of conformational isomers of nucleic acids, and to agarose compositions useful in such methods. The methods are distinguished by their ability to separate DNA by conformation as well as by molecular weight.
2. Description of the related art
It is well known to separate molecules according to their molecular weight by gel electrophoresis. The gels most commonly used for such purposes comprise agarose (a natural polysaccharide polymer) and cross-linked polyacrylamide. Traditionally, agarose gels have been used for the separation of larger molecules, such as double-stranded DNA with molecular sizes over 500 base pair (bp), while polyacrylamide gels (PAG) have been preferred for smaller molecules. Agaroses which are more sieving have become available. Nochumson, et al., Electrophoresis 81:213-218, (1981) showed that very-low melting temperature agarose such as "SeaPrep" agarose (a product of FMC Corporation, BioProducts Group, Philadelphia, Pa., U.S.A.) could be used for electrophoresis. Methods for making such agaroses are disclosed in U.S. Pat. No. 3,956,273--Guiseley, and reviewed by Guiseley in Industrial Polysaccharides, ed. M Yalpani, Elsevier, 1987; p. 139-147. In the latter publication, Guiseley shows that there is a linear relationship between the degree of substitution of an agarose and the reduction in gelling temperature (Tg) obtained by substitution. U.S. Pat. No. 4,319,975--Cook discloses that derivatized agarose gels have improved sieving capability compared to underivatized agarose of the same concentration. Other derivatized agaroses are described by Smith (U.S. Pat. No. 5,212,299), Shainoff (U.S. Pat. No. 4,275,196), and Nochumson (U.S. Pat. No. 4,504,641). U.S. Pat. No. 5,143,646--Nochumson, et al., discloses especially effective combinations of derivatized agarose (for sieving power) and partially depolymerized agarose (to control viscosity). An example of an agarose composition according to U.S. Pat. No. 5,143,646 is sold under the trademark "MetaPhor" by FMC Corporation, BioProducts Group, Philadelphia, Pa., U.S.A. This composition is useful as a gel for non-conformational electrophoretic separations and may comprise two hydrogels, at least one of which has been derivatized and independently at least one of which has been depolymerized sufficiently to reduce the casting-effective viscosity of the total gel composition, together with a resolving gel buffer. U.S. Pat. No. 5,143,646 further discloses that the degree of derivatization and to a lesser extent the concentration of the hydrocolloid (agarose) may be such that the gelling temperatures (Tg) are 10.degree. C.-25.degree. C., and further states that a Tg below about 10.degree. C. gives rise to weak or fragile gels which tend to crack whereas a Tg above 25.degree. C. falls off in sieving capacity (col 6). As described in FMC Corporation BioProducts Group technical bulletin P-18, MetaPhor agarose compositions can reliably separate DNA fragments differing in size by about 2%, or 4 base pairs, at 200 base pairs and higher, in non-denaturing systems. However, this degree of resolution is still not as discriminating as acrylamide gels can be. Stellwagen in Biochemistry 22:6186-6193, (1983) showed that in acrylamide gels, but not in agarose gels (p. 6186), fragments of DNA can migrate differently depending on their intrinsic conformation. In further work, Stellwagen in Biopolymers 30:309-324, (1990) indicated that the migration of a pair of DNA fragments, both having 147 base pairs, shows this effect clearly in standard polyacrylamide gels. The article states that " . . . pore size alone is not responsible for the anomalous electrophoretic mobilities . . . in polyacrylamide gels . . . " (p.323, last paragraph), and further states that the two fragments differed in their particular conformation, with one of the conformational isomers being "bent", and therefore retarded in its migration. Why the effect occurred in polyacrylamide but not agarose remains unclear.